EP2861264B1 - Kontrastmittel für fotoakustische bildgebung - Google Patents
Kontrastmittel für fotoakustische bildgebung Download PDFInfo
- Publication number
- EP2861264B1 EP2861264B1 EP13750954.3A EP13750954A EP2861264B1 EP 2861264 B1 EP2861264 B1 EP 2861264B1 EP 13750954 A EP13750954 A EP 13750954A EP 2861264 B1 EP2861264 B1 EP 2861264B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- icg
- hsa
- contrast agent
- formula
- albumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/225—Microparticles, microcapsules
Definitions
- the present invention relates to a contrast agent for photoacoustic imaging.
- a photoacoustic tomography (hereinafter, also referred to as "PAT") apparatus is known as one of apparatuses for visualizing in-vivo information.
- PAT apparatus an image can be obtained by measuring the intensity and the time of generation of a photoacoustic signal emitted from a substance (optical absorber) that absorbs light in an object to be measured when the object is irradiated with light, and computing a distribution of the substance in the object.
- ICG indocyanine green
- PAT apparatuses any substance that absorbs light and emits an acoustic wave in a living body may be used as an optical absorber.
- a blood vessel or a malignancy in the human body may be used as an optical absorber.
- molecules of indocyanine green hereinafter, also abbreviated as "ICG"
- ICG may be administered into the body and used as contrast agents.
- ICG well absorbs light in the near-infrared wavelength region, the light having little influence on the human body when the human body is irradiated with the light and having a high permeability to a living body.
- ICG may be used as a contrast agent in PAT apparatuses.
- ICG indicates a compound represented by formula (1) described below.
- the counter ion may not be Na+. Any counter ion, e.g., H+ or K+, may be used.
- NPL 1 reports a case of photoacoustic imaging of cerebral blood vessels of a rat with free ICG. According to this report, the photoacoustic signal intensity is reduced to a level equal to that of blood several tens of minutes after free ICG is administered in blood. This suggests that the administered substance is rapidly cleared from blood after administration.
- in vivo spectral fluorescence imaging is carried out to compare galactosyl human serum albumin (hGSA)-NMP1 with hGSA-IR800 and hGSA-ICG in terms of comparative fluorescence signal and the ability to detect peritoneal ovarian cancer metastases in mice models.
- hGSA galactosyl human serum albumin
- US 2011/294987 discloses a contrast agent for photoacoustic imaging comprising a particle, which is conjugated to a single-chain antibody, which is itself conjugated to an organic dye.
- aspects of the present invention provide a contrast agent for photoacoustic imaging, the contrast agent exhibiting high tumor accumulation and high photoacoustic signal intensity even when time has passed since administration.
- a contrast agent for photoacoustic imaging according to claim 1 is provided according to an aspect of the present invention.
- a contrast agent for photoacoustic imaging according to claim 3 is provided according to another aspect of the present invention.
- a contrast agent for photoacoustic imaging contains a complex including albumin bound to an organic dye, such as ICG, the dye absorbing light in the near-infrared wavelength region; hence, the contrast agent exhibits high accumulation in a tumor and high intensity of a photoacoustic signal emitted from the tumor, compared with the case where free ICG is administered.
- an organic dye such as ICG
- a contrast agent for photoacoustic imaging (hereinafter, also abbreviated as "PAI") according to an embodiment of the present invention will be described below.
- the contrast agent for PAI includes a complex including albumin covalently bound to an organic dye that absorbs light in the near-infrared wavelength region (hereinafter, also abbreviated as a "near-infrared absorbing organic dye").
- an organic dye that absorbs light in the near-infrared wavelength region hereinafter, also abbreviated as a "near-infrared absorbing organic dye”
- light in the near-infrared wavelength region refers to light having a wavelength of 600 nm to 1300 nm.
- a near-infrared absorbing organic dye such as ICG
- the dye is easily adsorbed on protein in blood and excreted from the body.
- the near-infrared absorbing organic dye may react with water molecules in blood to decompose.
- the dye exhibits short retention in blood and low accumulation in a tumor. Accordingly, in the case where a free near-infrared absorbing organic dye is used as a contrast agent for photoacoustic imaging, the intensity of the photoacoustic signal emitted from a tumor is low.
- albumin inhibits the adsorption of protein in blood on the near-infrared absorbing organic dye because the near-infrared absorbing organic dye is covalently bound to albumin.
- the contrast agent for PAI according to this embodiment is administered in blood of a living body, the contrast agent does not easily adsorb on protein in blood and is not easily excreted from the body.
- water molecules in blood do not easily approach the near-infrared absorbing organic dye because the near-infrared absorbing organic dye and albumin are covalently bound together; hence, the near-infrared absorbing organic dye is not easily decomposed.
- albumin has a half-life of about 20 days or less and is thus stable in a living body.
- the retention in blood should be improved, compared with the case where a free near-infrared absorbing organic dye is administered.
- the contrast agent for PAI according to this embodiment exhibits longer retention in blood and high accumulation in a tumor, compared with the case of administration of a free near-infrared absorbing organic dye.
- the effect of increasing the intensity of a photoacoustic signal emitted from the tumor should be provided.
- At least one albumin and at least one near-infrared absorbing organic dye may be covalently bound together.
- a plurality of albumins and a plurality of near-infrared absorbing organic dyes may be covalently bound together.
- at least one near-infrared absorbing organic dye may be covalently bound to albumin, and the remaining near-infrared absorbing organic dyes may be noncovalently bound.
- the complex includes a near-infrared absorbing organic dye and a plurality of albumins
- at least one albumin may be covalently bound to the near-infrared absorbing organic dye, and the remaining albumins may be noncovalently bound.
- albumin and the near-infrared absorbing organic dye are covalently bound together
- a site this may also be referred to as a "group” of albumin with a portion (typically, H or OH) of albumin removed and a site of the near-infrared absorbing organic dye with a portion (typically, H or OH) of the near-infrared absorbing organic dye removed are covalently bound.
- the "complex” can also be referred to as a "molecule" from another point of view.
- the number of the near-infrared absorbing organic dyes covalently bound to one albumin is referred to as a "dye labeling index".
- the dye labeling index is 1.6 or more.
- the dye labeling index may be lower than 3.1.
- the dye labeling index is preferably 1.6 or more and 3.0 or less. The reason for this is that when the dye labeling index falls within the range described above, high tumor accumulation is obtained.
- the dye labeling index was calculated by measuring concentrations of the near-infrared absorbing organic dye and albumin in a sample and determining the ratio of the concentration of the near-infrared absorbing organic dye to the concentration of albumin (the concentration of the near-infrared absorbing organic dye/the concentration of albumin).
- the concentration of the near-infrared absorbing organic dye was calculated from the absorbance at a specific absorption wavelength of the dye. For example, when ICG-Sulfo-OSu (a compound represented by formula (2) described below) is used, a
- wavelength of 800 nm may be used as the specific absorption wavelength.
- a wavelength of 750 nm may be used as the specific absorption wavelength.
- another specific absorption wavelength may be used.
- the concentration of albumin may be determined by, for example, the BCA assay.
- the contrast agent for PAI may contain a complex represented by formula (I),
- A represents a site of albumin with one amino group removed.
- A' represents formula (i) or (ii).
- "*" in each of formulae (i) and (ii) is bound to a nitrogen atom (N) in formula (I).
- Z's each represent a hydrogen atom, a sulfonic group, or a cyclic aromatic ring selected from the group consisting of a benz[e]indole ring, a benz[f]indole ring, and a benz[g]indole ring formed together with an indole ring bound to a corresponding one of Z's.
- the hydrogen atoms of the cyclic aromatic ring each may be replaced with an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or a sulfonic group.
- R 1 's each represent an alkyl group having 1 to 10 carbon atoms or -(CH 2 ) b -SO 3 - (wherein b represents an integer of 1 to 10).
- a halide ion e.g., a chloride ion, a bromide ion, or an iodide ion
- an organic acid ion e.g., an acetate ion, a tartrate ion, or a succinate ion
- a counter ion e.g., a halide ion, e.g., a chloride ion, a bromide ion, or an iodide ion
- an organic acid ion e.g., an acetate ion, a tartrate ion, or a succinate ion
- R 2 's and R 3 's each independently represent a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, -(CH 2 ) b -SO 3 - (wherein b represents an integer of 1 to 10), or -(CH 2 ) b -SO 3 X (wherein b represents an integer of 1 to 10, and X represents sodium, potassium, ammonium, triethylammonium, lysine, or arginine).
- a's each represent an integer of 1 to 10
- n's each represent 2 or 3.
- R 4 represents an alkyl group having 1 to 10 carbon atoms or -(CH 2 ) b -SO 3 X (wherein b represents an integer of 1 to 10, and X represents sodium, potassium, ammonium, triethylammonium, lysine, or arginine).
- a portion indicated by A-N-H corresponds to the site of albumin with a portion of albumin removed.
- A' corresponds to the site of the near-infrared absorbing organic dye with a portion of the near-infrared absorbing organic dye removed.
- formula (i) may be represented by any one of formulae (i-1) to (i-6) described below.
- Y- represents a halide ion, e.g., a chloride ion, a bromide ion, or an iodide ion, or an organic acid ion, e.g., an acetate ion, a tartrate ion, or a succinate ion.
- halide ion e.g., a chloride ion, a bromide ion, or an iodide ion
- organic acid ion e.g., an acetate ion, a tartrate ion, or a succinate ion.
- X's each represent sodium, potassium, ammonium, triethylammonium, lysine, or arginine.
- formula (ii) may be represented by formula (ii-1) or (ii-2) described below.
- X's each represent sodium, potassium, ammonium, triethylammonium, lysine, or arginine.
- a's may each represent an integer of 2 to 6.
- b's in R 1 's, R 2 's, and R 3 's may each represent an integer of 2 to 6.
- the complex may be represented by formula (I-1) described below.
- A represents a site of albumin with one amino group of albumin removed.
- the contrast agent for PAI may contain a capture molecule that binds specifically to a target site.
- Albumin according to this embodiment is an abundant protein in blood (35 to 50 g/ L), the protein having a molecular mass of 66.5 kDa and containing 585 amino acids in its complete sequence. Albumin is localized in vivo to play many roles, such as osmotic control.
- human serum albumin (HSA) and bovine serum albumin (BSA) may be used as albumin according to this embodiment.
- a variant of HSA or BSA may also be used.
- a fragment thereof may also be used.
- HSA, a variant of HSA, a fragment of HSA, or a fragment of a variant of HSA, which is believed to be safe for the human body may be used.
- Albumin according to this embodiment may be an extract from human blood or a product from Escherichia coli or the like.
- Albumin according to this embodiment has a homology of at least 95% or more, as compared with the complete sequence or a partial sequence from the complete sequence of HSA.
- Albumin has a plurality of lysine residues or a free cysteine residue at positions where the near-infrared absorbing organic dye is accessible.
- a chemical bond between albumin and the near-infrared absorbing organic dye is formed, for example, an amide bond between an amino group of a lysine residue of albumin and a carboxy group of the near-infrared absorbing organic dye is exemplified.
- the near-infrared absorbing organic dye is represented by formula (II) described below. [Chem.18] B-B' (II)
- B represents formula (i) or (ii) described above.
- B' represents any one of formulae (iii) to (vi) described below.
- a compound represented by formula (2) (ICG-Sulfo-OSu (registered trademark, manufactured by Dojindo Laboratories)), a compound represented by formula (3), a compound represented by formula (4), a compound represented by formula (5), a compound represented by formula (6), or a compound represented by formula (7) may be used.
- albumin and the near-infrared absorbing organic dye may be covalently bound together by a known coupling reaction with an amino group, a thiol group, a carboxy group, or a hydroxy group provided therebetween.
- a plurality of amino groups are present in albumin and react efficiently and selectively in a weak alkaline pH region.
- the near-infrared absorbing organic dye bound to albumin by the reaction may be washed and purified by a known protein purification method, for example, ultrafiltration or size-exclusion column chromatography.
- albumin and the near-infrared absorbing organic dye With respect to the bond between albumin and the near-infrared absorbing organic dye, the amino group, the thiol group, the carboxy group, or the hydroxy group present on the surface of albumin may be directly bound to a derivative of the near-infrared absorbing organic dye. Alternatively, albumin and the near-infrared absorbing organic dye may be bound together with any of a variety of cross-linkers.
- the complex may be in the form of particles.
- Each of the particles may be any of shapes, such as spherical, elliptic, planar, and one-dimensional string-like shapes.
- the hydrodynamic average particle size (hereinafter, abbreviated simply as "particle size", in some cases) may be less than 1000 nm when measured by a dynamic light scattering method. A particle size of less than 1000 nm results in the accumulation of a large number of particles in a tumor site by the enhanced permeability and retention (EPR) effect, compared with normal sites in a living body.
- EPR enhanced permeability and retention
- the contrast agent for PAI accumulated in the tumor site results in the specific visualization of the tumor site by the use of a photoacoustic imaging apparatus.
- the particle size of the particles is preferably 200 nm or less and more preferably 50 nm or less when measured by the dynamic light scattering method. The reason for this is presumably that when the particle size of the particles is 200 nm or less, the contrast agent for PAI according to this embodiment is less likely to be taken up by macrophages in blood, thereby increasing the retention in blood. In addition, when the particle size of the particles is 50 nm or less, the tissue permeability of the particles should be increased, thereby increasing the accumulation of the particles that have reached a target site.
- the particle size of the particles may be determined by measuring the hydrodynamic average particle size using the dynamic light scattering (DLS) method with a dynamic light scattering spectrophotometer (DLS-8000, manufactured by Otsuka Electronics Co., Ltd).
- DLS dynamic light scattering
- the contrast agent for PAI may contain a dispersion medium in addition to the complex.
- the PAI is a concept including photoacoustic tomography.
- the dispersion medium include physiological saline, distilled water for injection, phosphate-buffered saline, and an aqueous glucose solution.
- the contrast agent for PAI according to this embodiment may contain a pharmaceutically acceptable additive, such as a vasodilator, as needed.
- the particles may be dispersed in the dispersion medium in advance.
- the particles and the dispersion medium may be prepared as a kit, and the particles may be dispersed in the dispersion medium prior to the administration of the contrast agent into a living body.
- the contrast agent for PAI in the contrast agent for PAI according to this embodiment, a larger number of the particles can be accumulated in a tumor site than in normal sites in a living body by the EPR effect when the contrast agent is administered into a living body.
- the intensity of an acoustic wave emitted from the tumor site can be increased, compared with the intensity of acoustic waves emitted from the normal sites.
- the contrast agent for PAI according to this embodiment may be used for tumor imaging.
- the contrast agent for PAI according to this embodiment may also be used to image a lymph node.
- the contrast agent may be used as a contrast agent for a sentinel lymph node (hereinafter, abbreviated as "SLN", in some cases).
- SSN sentinel lymph node
- a near-infrared absorbing organic dye such as ICG
- ICG an intracranial pressure
- the contrast agent for PAI according to this embodiment has a larger molecular size than the near-infrared absorbing organic dye, thus reducing the rate of diffusion in tissues. As a result, the retention time in the sentinel lymph node should be extended.
- the contrast agent for PAI according to this embodiment may be used to image a lymph node, in particular, a sentinel lymph node.
- the capture molecule in this embodiment is, for example, a substance that binds specifically to a target site, such as a tumor, or a substance that binds specifically to a substance present around a target site.
- the capture molecule may be freely selected from biomolecules and chemical substances, such as pharmaceuticals. Specific examples thereof include proteins, antibodies, antibody fragments, enzymes, biologically active peptides, glycopeptides, sugar chains, lipids, and molecule-recognizing compounds. These substances may be used separately or in combination.
- the use of the particles to which the capture molecules are chemically bonded enables the specific detection of a target site and the tracing of the dynamics, localization, efficacy of medicine, metabolism, and so forth of the target substance.
- protein refers to a compound in which 90 or more natural or non-natural amino acids are connected by amide bonds.
- polypeptide refers to a compound in which 30 or more and less than 90 natural or non-natural amino acids are connected by amide bonds.
- peptide refers to a compound in which less than 30 natural or non-natural amino acids are connected by amide bonds.
- protein, polypeptide, and peptide are classified by the number of amino acids connected, regardless of the presence or absence of various modifications.
- the capture molecule may be protein, polypeptide, or peptide.
- the capture molecule may be an antibody, which is a protein.
- the capture molecule may be a single-chain antibody.
- contrast agent for photoacoustic imaging, in which the contrast agent comprises a complex which is represented by formula (III): [Chem.29] ALB-L-C (III)
- ALB represents the albumin which is covalently bound by the organic dye
- C represents a capture molecule
- L represents a linker, ALB being bound to L, and L being bound to C.
- the capture molecule may be a protein, a polypeptide, or a peptide.
- the protein may be a single-chain antibody.
- L in formula (III) may include one or more succinimidyl groups at an end and one or more maleimido groups at the other end.
- L in formula (III) may include one or more succinimidyl groups at the end, one or more maleimido groups at the other end, and one or more ethylene glycol moieties.
- L in formula (III) may represent succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester or SUNBRIGHT MA-100TS.
- L in formula (III) may represent succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
- L in formula (III) may include two or more succinimidyl groups at an end.
- L in formula (III) may include two or more succinimidyl groups at an end and one or more ethylene glycol moieties.
- L in formula (III) may represent bis-N-succinimidyl-(pentaethylene glycol) ester.
- the contrast agent for photoacoustic imaging according to the present embodiment may contain an addition agent used in freeze-drying.
- addition agents include glucose, lactose, mannitol, polyethylene glycol, glycine, sodium chloride, and sodium hydrogen phosphate.
- One type of addition agent may be used alone or some types may be used in combination.
- a method for detecting the contrast agent for PAI according to this embodiment, the contrast agent being administered into a living body, with a photoacoustic imaging apparatus will be described below.
- the method for detecting the contrast agent for PAI according to this embodiment includes steps (a) and (b) described below.
- the photoacoustic imaging method according to this embodiment may include a step other than the following steps:
- the photoacoustic imaging method may include a step of reconstituting a spatial photoacoustic signal intensity distribution on the basis of the wavelength, the phase, the time information, and so forth of the resulting acoustic wave obtained in step (b).
- Three-dimensional image reconstruction may be performed on the basis of the wavelength, the phase, and the time information of the acoustic wave obtained in step (b).
- Data obtained by the image reconstruction may be in any form as long as the positional information on the photoacoustic signal intensity distribution can be determined.
- the photoacoustic signal intensity may be expressed in three-dimensional space or on a two-dimensional surface.
- the information of the same observation object is acquired by another imaging method, and the positional relationship between the information and the photoacoustic signal intensity distribution can be acquired.
- step (a) described above a subject to which the contrast agent for PAI according to this embodiment is administered by a method, for example, oral administration or injection may be used.
- an apparatus configured to generate light with which the subject is irradiated and an apparatus configured to detect an acoustic wave emitted from the contrast agent for PAI according to this embodiment are not particularly limited.
- any light source may be used without limitation as long as the subject can be irradiated with pulsed laser light having at least one wavelength selected from the range of 600 nm to 1300 nm.
- the apparatus configured to emit pulsed laser light for example, a titanium-sapphire laser (LT-2211-PC, manufactured by Lotis Ltd.), OPO laser (LT-2214 OPO, manufactured by Lotis Ltd.), or an alexandrite laser may be used.
- the apparatus configured to detect an acoustic wave is not particularly limited, and any of a variety of apparatuses may be used.
- a commercially available photoacoustic imaging apparatus (Nexus 128, manufactured by Endra Inc.) may be used.
- a target site for example, a tumor, a lymph node, or a blood vessel, can be imaged through steps (a) and (b).
- the photoacoustic signal intensity was measured as described below.
- a commercially available photoacoustic imaging apparatus (Nexus128, manufactured by Endra Inc.) was used. Photoacoustic signals were measured at predetermined timings before and after the administration of a prepared contrast agent for PAI, and three-dimensional reconstruction data was acquired for each timing. The photoacoustic signal intensity in a region of interest (ROI) was measured on the basis of the resulting three-dimensional reconstruction data using software (GEHC MICROVIEW, GE Healthcare) or the like.
- the dye labeling index to albumin was calculated by measuring the absorbance of a sample.
- a dye concentration in the sample was calculated from absorbance at a specific absorption wavelength of the dye used. Specifically, when ICG-Sulfo-OSu (compound represented by formula (2) described above) was used, the absorbance was measured at 800 nm. When the compound represented by formula (5) described above was used, the absorbance was measured at 750 nm.
- the concentration of ICG was determined by diluting the sample with 5% SDS, measuring the absorbance, and calculating the concentration of ICG from a previously formed calibration curve of the dye in SDS.
- the concentration of HSA was calculated by the BCA assay.
- the evaluation of the contrast agent for PAI into a tumor mass was performed using tumor-bearing mice.
- the tumor-bearing mice were prepared by subcutaneously implanting a human gastric cancer cell line (N87) or a human cervical cancer cell line (HeLa) into nude mice. Then 130 nmol of a contrast agent in terms of the amount of the dye was administered into each of the tumor-bearing mice.
- Photoacoustic imaging was performed 5 minutes after the administration and 1 day after the administration.
- fluorescence imaging of the tumor-bearing mice was performed 1 day after the administration. Fluorescence imaging was performed with IVIS (registered trademark) Imaging System to measure fluorescence intensity in a region of interest (ROI) of the tumor site.
- IVIS registered trademark
- the transfer of the contrast agent for PAI into a sentinel lymph node was evaluated by the use of a mouse popliteal lymph node. Ten microliters of the contrast agent was administered subcutaneously into the plantar surface of a nude mouse. Photoacoustic imaging of the mouse popliteal lymph node was performed after 1 day.
- mouse popliteal lymph nodes were removed 1 day after the administration of various contrast agents, homogenized, and extracted. The resulting extracts were subjected to absorbance measurement.
- the accumulation ratios of various materials were calculated when the accumulation 1 day after the administration of ICG was defined as 1.
- a compound according to this embodiment is a compound in which an amino group on the surface of HSA is covalently bound to a near-infrared absorbing organic dye.
- a typical structure of ICG-HSA is represented by formula (IV).
- a typical structure of ICG'-HSA is represented by formula (V).
- HSA Human serum albumin
- bicarbonate buffer solutions pH: 9.4
- DMSO dimethyl sulfoxide
- the dye labeling index can be changed in any range by changing the feed molar ratio of the dye derivative to HSA.
- the filtrates obtained by sterile filtration did not exhibit absorbance at the specific absorption wavelength of the dye; hence, the dye labeling index was not calculated.
- ICG-HSA (1) and ICG-HSA (2) are reference example outside the scope of the invention as claimed [Table 2] Sample name Feed molar ratio (compound of formula 5:HSA) Dye labeling index ICG'-HSA (1) 1:1 0.6 ICG'-HSA (3) 3:1 1.2 ICG'-HSA (9) 9:1 2.0 ICG'-HSA (24) 24:1 2.7 ICG'-HSA (1) and ICG'-HSA (3) are reference example outside the scope of the invention as claimed
- the hydrodynamic average particle size of ICG-HSA (7) prepared by the foregoing method was measured with a dynamic light scattering spectrophotometer (DLS-8000, manufactured by Otsuka Electronics Co., Ltd). Table 3 describes the results.
- the average particle sizes of ICG available from Pharmaceutical and Medical Device Regulatory Science of Japan
- HSA HSA
- ICG-HSA (7)- encapsulating liposome particles ICG-encapsulating micellar particles are also described.
- the ICG-HSA (7)-encapsulating liposome particles refer to particles in which ICG-HSA (7) prepared by the foregoing method is encapsulated in phospholipid liposome by a known method.
- the ICG-encapsulating micellar particles refer to micellar particles in which ICG is contained in surfactant micelles by a known emulsification technique.
- Table 4 demonstrates that the photoacoustic signal intensity at the tumor site increased markedly immediately after (5 minutes after) the administration of ICG-HSA (7) and that the photoacoustic signal intensity immediately after the administration was maintained even 1 day after the administration. [Table 4] Relative photoacoustic signal intensity Before administration 1.0 Five minutes after administration 7.2 One day after administration 55
- the accumulation in a sentinel lymph node was evaluated by photoacoustic imaging.
- 130 nmol of ICG-HSA (7) in terms of the amount of the dye was administered subcutaneously into the plantar surface of a nude mouse.
- an evaluation was made as to whether the sentinel lymph node was visualized 1 day after the administration by photoacoustic imaging.
- Table 6 and Fig. 2 illustrate the results.
- substantially no photoacoustic signal was observed from the sentinel lymph nodes 1 day after the administration, so that the sentinel lymph nodes were not visualized.
- the SLN was clearly visualized (see Fig.
- ICG-HSA (7) had visualization properties substantially the same as ICG-HSA-encapsulating liposome.
- the SLN was removed.
- the removed SLN and an SLN into which no contrast agent was administered were juxtaposed to each other and subjected to photoacoustic imaging.
- Fig. 3 illustrates the results. A significant photoacoustic signal was observed from the SLN 1 day after the administration of ICG-HSA (7), as compared with the unadministered SLN.
- Aqueous solutions of ICG-HSA (2), ICG-HSA (7), ICG-HSA (21), and ICG-HSA (50) were prepared by the method described in EXAMPLE.
- Tumor model mice were prepared by subcutaneously implanting Colon 26 cells into BALB/c Slc-nu/nu mice.
- the aqueous ICG-HSA solutions were intravenously injected in volumes of 100 microliters each (13 nmol in terms of ICG) into the tumor model mice to evaluate the tumor accumulation.
- ICG-Gly a compound was synthesized by the reaction of ICG-Sulfo-OSu with glycine in a molar ratio of 1:1 (hereinafter, abbreviated as "ICG-Gly") and used as a control sample.
- mice were euthanized with carbon dioxide 24 hours after administration.
- the tumor tissues were removed and transferred to plastic tubes.
- An aqueous solution of 1% Triton X-100 was added to each of the tubes in an amount 1.25 times the weight of the tumor tissues.
- Each of the resulting mixtures was homogenized with a plastic pestle.
- DMSO was added to each mixture in an amount 20.25 times the weight of the tumor tissues to prepare a solution of the dye extracted from the tumor tissues.
- An aqueous ICG-HSA solution having a known concentration and an aqueous ICG-Gly solution serving as a control were diluted with the solution of the tumor tissues in Triton X-100 to various concentrations.
- DMSO was added to each of the resulting dilute solutions in an amount 20.25 times the amount of each dilute solution to prepare standard solutions for calibration.
- the fluorescence intensity of the solutions of the dye extracted from the tumor tissues and the standard solutions for calibration was measured with the solutions in the tubes using IVIS (registered trademark) Imaging System 200 Series (XENOGEN Corporation) to quantitatively determine the amount of the dye (%ID/g) in the tumor tissues.
- Aqueous solutions of ICG-HSA (2), ICG-HSA (7), ICG-HSA (21), and ICG-HSA (50) were prepared by the method described in EXAMPLE.
- Tumor model mice were prepared by subcutaneously implanting Colon 26 cells into BALB/c Slc-nu/nu mice.
- the aqueous ICG-HSA solutions were intravenously injected in volumes of 100 microliters each (13 nmol in terms of ICG) into the tumor model mice to evaluate the tumor accumulation.
- an aqueous solution of ICG available from Pharmaceutical and Medical Device Regulatory Science of Japan was used as a control sample.
- mice were euthanized with carbon dioxide 24 hours after administration.
- the tumor tissues were removed and transferred to plastic tubes.
- An aqueous solution of 1% Triton X-100 was added to each of the tubes in an amount 1.25 times the weight of the tumor tissues.
- Each of the resulting mixtures was homogenized with a plastic pestle.
- DMSO was added to each mixture in an amount 20.25 times the weight of the tumor tissues to prepare a solution of the dye extracted from the tumor tissues.
- An aqueous ICG-HSA solution having a known concentration and an aqueous ICG solution were diluted with the solution of the tumor tissues in Triton X-100 to various concentrations.
- DMSO was added to each of the resulting dilute solutions in an amount 20.25 times the amount of each dilute solution to prepare standard solutions for calibration.
- the fluorescence intensity of the solutions of the dye extracted from the tumor tissues and the standard solutions for calibration was measured with the solutions in the tubes using IVIS (registered trademark) Imaging System 200 Series (XENOGEN Corporation) to quantitatively determine the amount of the dye (%ID/g) in the tumor tissues.
- Table 8 describes the accumulation in the Colon 26 cell mass 24 hours after the administration of the aqueous ICG-HSA solutions and the control.
- the results suggested that the tumor accumulation was improved by covalently binding the dye to HSA, compared with the aqueous ICG solution serving as a control sample.
- ICG-HSA (7) and ICG-HSA (15) exhibited high values of the ratio of tumor accumulation to blood accumulation 24 hours after the administration. This suggested that they were specifically accumulated in tumor, compared with blood.
- ICG-HSA (7) also exhibited high photoacoustic signal intensity at the tumor site 1 day after the administration. Among these samples, thus, ICG-HSA (7) was most effective in visualizing the tumor.
- a gene fragment encoding a single-chain antibody (scFv) moiety was prepared on the basis of the gene sequence of the variable region of IgG binding to HER2.
- a 6 x His tag comprising six consecutive histidine residues for protein purification was bound to the C-terminus of the prepared gene.
- two glycine residues serving as a spacer and a cysteine residue to introduce a signal generating molecule were arranged downstream thereof (SEQ. ID. NO: 1).
- a plasmid pET-22b (+) (Novagen) in which the foregoing gene fragment was inserted downstream of the T7 promoter was introduced into Escherichia coli BL21 (DE3) to give a strain for expression.
- the solution subjected to salting out was allowed to stand overnight at 4 degrees Celsius and centrifuged at 8000 x g for 30 minutes at 4 degrees Celsius to collect precipitates.
- the resulting precipitates were dissolved in 20 mM Tris HCl/500 mM NaCl buffer. The mixture was dialyzed against 1 L of the buffer. After the dialysis, the protein solution was added to a column filled with His Bind (registered trademark) Resin (Novagen) and purified by metal chelate affinity chromatography using a Ni ion.
- Human serum albumin (albumin from human serum: HSA, SIGMA) was dissolved in a bicarbonate buffer solution (pH: 8.5) in a concentration of 10 mg/mL to prepare an HSA solution. Then 1 mg of an ICG derivative (ICG-Sulfo-Osu, Dojindo Laboratories) was dissolved in 0.1 mL of DMSO. The resulting DMSO solution was added to the HSA solution in an amount 7 times the molar amount of HSA. The reaction mixture was allowed to stand at 37 degrees Celsius for 2 hours. The reaction mixture was subjected to ultrafiltration (30 kDa) to remove unreacted substances, thereby preparing an aqueous solution of ICG-HSA.
- Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC, PIERCE) was added thereto in an amount 60 times the molar amount of ICG-HSA.
- the mixture was allowed to stand at 4 degrees Celsius for 1 hour.
- the resulting reaction mixture was subjected to gel filtration (PD-10), so that the replacement with a phosphate buffer solution (PBS) and the separation of unreacted substances were performed, thereby preparing an aqueous solution of Sulfo-SMCC-modified ICG-HSA.
- PBS phosphate buffer solution
- Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride was added to the single-chain scFv in an amount 14 times the molar amount of the single-chain antibody, scFv. The mixture was reacted at room temperature for 2 hours. Then scFv was mixed with Sulfo-SMCC-modified ICG-HSA prepared as described above in an amount 1 or 2 times the molar amount of Sulfo-SMCC-modified ICG-HSA. The mixture was reacted at room temperature for 5 hours.
- scFv-ICG-HSA scFv-ICG-HSA
- a compound prepared by the reaction of scFv in an amount 1 time the molar amount of Sulfo-SMCC-modified ICG-HSA is referred to as "scFv-ICG-HSA-1”.
- a compound prepared by the reaction of scFv in an amount 2 times the molar amount of Sulfo-SMCC-modified ICG-HSA is referred to as "scFv-ICG-HSA-2".
- Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC, PIERCE) was added to HSA in an amount 60 times the molar amount of HSA. The mixture was allowed to stand at 4 degrees Celsius for 1 hour. The resulting reaction mixture was subjected to gel filtration (PD-10), so that the replacement with a phosphate buffer solution (PBS) and the separation of unreacted substances were performed, thereby preparing an aqueous solution of Sulfo-SMCC-modified HSA.
- PBS phosphate buffer solution
- Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride was added to the single-chain scFv in an amount 14 times the molar amount of the single-chain scFv. The mixture was reacted at room temperature for 2 hours. Then scFv was mixed with Sulfo-SMCC-modified HSA prepared as described above in an amount 3 times the molar amount of Sulfo-SMCC-modified HSA. The mixture was reacted at room temperature for 5 hours. The reaction mixture was subjected to ultrafiltration (50 kDa) to remove unreacted substances, thereby providing scFv-immobilized HSA (scFv-HSA).
- ICG was added to scFv-HSA prepared as described above in an amount 7, 21, or 70 times the molar amount of scFv-HSA.
- the mixture was reacted for 2 hours and subjected to ultrafiltration (30 kDa) to remove unreacted substances, thereby preparing ICG-labeled scFv-HSA (scFv-HSA-ICG).
- Compounds prepared by the reaction of ICG in amounts 7, 21, and 70 times the molar amount of scFv-HSA are referred to as scFv-HSA-ICG-7, scFv-HSA-ICG-21, and scFv-HSA-ICG-70, respectively.
- the dye labeling indices to HSA were calculated.
- the dye labeling indices were calculated by the quantitative determination of protein using the BCA assay and by the measurement of the concentrations on the basis of the absorbance of ICG. Table 9 describes the results.
- scFv-ICG-HSA and scFv-HSA prepared as described above the number of single-chain antibody molecules (scFv's) immobilized to HSA was calculated. Table 9 describes the results. The number of scFv's immobilized was calculated by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and measuring Coomassie staining intensity of bands.
- HER2 which is an antigen
- scFv-ICG-HSA and scFv-HSA-ICG complexes prepared as described above were measured with a Biacore X System (GE Healthcare Corp.) to measure HER2-binding capacities.
- the antigen Recombinant Human ErbB2/Fc Chimera (R&D Systems, Inc.) was used.
- the antigen was immobilized by amine coupling to a carboxymethyldextran chain on a surface of Chip CM5 according to the manufacturer's recommendation. The amount of the antigen immobilized was about 5000 RU.
- PBS-T (2.68 mM KCl/137 mM NaCl/ 1.47 mM KH 2 PO 4 /1 mM Na 2 HPO 4 / 0.005% Tween 20, pH: 7.4) was used as a running buffer. Concentrations of samples were set to 100 nM to 800 nM. The samples were injected at a flow rate of 20 microliters per minutes to evaluate HER2-binding capacities (dissociation constants K D [M]). Table 9 describes the results. The results demonstrated that a larger number of scFv's immobilized resulted in a higher HER2-binding capacity.
- HER2-specific tumor accumulation of the molecular probes (ICG-HSA, scFv-ICG-HSA, and scFv-HSA-ICG) prepared as described above, an experiment was performed as described below.
- the molecular probes were administered to tumor-bearing model mice into which the HER2-positive N87 cell line and the HER2-negative SUIT-2 cell line were implanted. The tumors were removed 1 day after the administration. To the tumors, 1% Triton X-100 was added. The mixtures were homogenized. Dimethyl sulfoxide (DMSO) was added to each resulting homogenate in an amount 9 times the amount of the homogenate, thereby preparing a solution.
- DMSO Dimethyl sulfoxide
- the fluorescence intensity of these solutions was measured to calculate the tumor accumulation [%ID/g] per tumor weight of the molecular probes administered to the mice. Furthermore, a value obtained by dividing the tumor accumulation in N87 by the tumor accumulation in SUIT-2 (N87/SUIT-2) was calculated as a value indicating HER2 specificity. Table 9 describes the results. The results demonstrated that scFv-ICG-HSA-1, scFv-ICG-HSA-2, and scFv-HSA-ICG-7 exhibited HER2-specific accumulation.
- Comparison of scFv-HSA-ICG-7, scFv-HSA-ICG-21, and scFv-HSA-ICG-70 revealed that when the dye labeling index was 6.6 or more, HER2-specific accumulation was not observed.
- Sample name Number of scFv's immobilized Dye labeling index K D to HER2 [M] Tumor accumulation (after 1 day) [%ID/g] HER2 specificity N87 SUIT-2 N87/SUIT-2 scFv-ICG-HSA-1 0.69 1.9 5.9E-08 1.4 0.9 1.6 scFv-ICG-HSA-2 1.7 1.9 2.5E-08 2.3 1.1 2.1 scFv-HSA-7 2.9 2.0 1.3E-08 1.9 0.6 3.2 scFv-HSA-21 2.9 6.6 1.4E-08 0.6 0.5 1.2 scFv-HSA-70 2.9 21 8.5E-08 0.8 0.7 1.1 ICG-HSA -
- Human serum albumin (albumin from human serum: HSA, SIGMA) was dissolved in a bicarbonate buffer solution (pH: 8.5) in a concentration of 10 mg/mL to prepare an HSA solution. Then 1 mg of an ICG derivative (ICG-Sulfo-Osu, Dojindo Laboratories) was dissolved in 0.1 mL of DMSO. The resulting DMSO solution was added to the HSA solution in an amount 7 times the molar amount of HSA. The reaction mixture was allowed to stand at 37 degrees Celsius for 2 hours. The reaction mixture was subjected to ultrafiltration (30 kDa) to remove unreacted substances, thereby preparing an aqueous solution of ICG-HSA.
- SM(PEG)2 succinimidyl[(N-maleimidopropionamido) -diethyleneglycol] ester, PIERCE
- SUNBRIGHT MA-100TS NOF CORPORATION
- the number of molecules of each linker attached to ICG-HSA was calculated from a change in the number of amino acids in ICG-HSA before and after the attachment of the linker.
- the number of amino acids was quantitatively determined by a color reaction with 2,4,6-trinitrobenzenesulfonic acid.
- Affibody (registered trademark, Affibody) is a polypeptide serving as a HER2 capture molecule.
- Dithiothreitol NACALAI TESQUE, INC.
- PD-10 gel filtration
- This aqueous solution was mixed with each of the aqueous solutions of ICG-HSA modified with two types of linkers in different ratios. The mixtures were stirred at 25 degrees Celsius for 2 hours or more. Then the mixtures were subjected to ultrafiltration (30kDa) to remove unreacted Affibody (registered trademark), thereby preparing Affibody (registered trademark)-immobilized ICG-HSA. The number of Affibody (registered trademark) molecules immobilized was calculated by quantitatively determining unfixed molecules eluted in the filtrate by the ultrafiltration.
- Affibody (registered trademark)-immobilized ICG-HSA prepared by the use of SM(PEG)2 as the linker in an amount 10 times the molar amount of ICG-HSA is referred to as L1-ICG-HSA.
- Affibody (registered trademark)-immobilized ICG-HSA prepared by the use of SM(PEG)2 as the linker in an amount 100 times the molar amount of ICG-HSA is referred to as L2-ICG-HSA.
- Affibody (registered trademark)-immobilized ICG-HSA prepared by the use of SUNBRIGHT MA-100TS as the linker in an amount 10 times the molar amount of ICG-HSA is referred to as L3-ICG-HSA.
- Affibody (registered trademark)-immobilized ICG-HSA prepared by the use of SUNBRIGHT MA-100TS as the linker in an amount 100 times the molar amount of ICG-HSA is referred to as L4-ICG-HSA.
- SPR surface plasmon resonance
- Human serum albumin (albumin from human serum: HSA, SIGMA) was dissolved in a bicarbonate buffer solution (pH: 8.5) in a concentration of 10 mg/mL to prepare an HSA solution. Then 1 mg of an ICG derivative (ICG-Sulfo-Osu, Dojindo Laboratories) was dissolved in 0.1 mL of DMSO. The resulting DMSO solution was added to the HSA solution in an amount 7 times the molar amount of HSA. The reaction mixture was allowed to stand at 37 degrees Celsius for 2 hours. The reaction mixture was subjected to ultrafiltration (30 kDa) to remove unreacted substances, thereby preparing an aqueous solution of ICG-HSA.
- the number of molecules of each linker attached to ICG-HSA was calculated from a change in the number of amino acids in ICG-HSA before and after the attachment of the linker.
- the number of amino acids was quantitatively determined by a color reaction with 2,4,6-trinitrobenzenesulfonic acid.
- the number of linker molecules in ICG-HSA modified with SM(PEG)2 (hereinafter, abbreviated as "SM-ICG-HSA") was 13.
- the number of linker molecules in ICG-HSA modified with SMCC (hereinafter, abbreviated as "CC-ICG-HSA”) was 9.
- the number of linker molecules in ICG-HSA modified with BS(PEG)5 was 15.
- scFv, Affibody (registered trademark), or HER2-binding peptides were immobilized to ICG-HSA modified with the three types of linkers described above.
- the sequences of the HER2-binding peptide were described below.
- scFv was subjected to reduction treatment with TCEP, and Affibody (registered trademark) was subjected to reduction treatment with dithiothreitol, in the same ways as described above in advance.
- One of the linker-modified ICG-HSA samples was mixed with the HER2-binding peptide, Affibody (registered trademark) subjected to reduction treatment, or scFv subjected to reduction treatment. The mixture was stirred at 25 degrees Celsius for 2 hours or more.
- the mixture was subjected to ultrafiltration (30 kDa) to remove unreacted HER2-binding molecules, thereby preparing HER2-binding-molecule-immobilized ICG-HSA.
- the number of the HER2-binding molecules immobilized was calculated by quantitatively determining unfixed molecules eluted in the filtrate by the ultrafiltration.
- BS-ICG-HSA to which the peptide of SEQ. ID. NO: 2 was immobilized is referred to as L5-ICG-HSA.
- SM-ICG-HSA to which the peptide of SEQ. ID. NO: 3 was immobilized is referred to as L6-ICG-HSA.
- L7-ICG-HSA L7-ICG-HSA
- BS-ICG-HSA to which the peptide of SEQ. ID. NO: 3 was immobilized is referred to as L8-ICG-HSA
- SM-ICG-HSA to which Affibody (registered trademark) was immobilized is referred to as L9-ICG-HSA
- CC-ICG-HSA to which Affibody (registered trademark) was immobilized is referred to as L10-ICG-HSA.
- BS-ICG-HSA to which Affibody (registered trademark) was immobilized is referred to as L11-ICG-HSA.
- scFv-immobilized SM-ICG-HSA is referred to as L12-ICG-HSA.
- scFv-immobilized CC-ICG-HSA is referred to as L13-ICG-HSA.
- scFv-immobilized BS-ICG-HSA is referred to as L14-ICG-HSA.
- SPR surface plasmon resonance
- mice Female outbred BALB/c Slc-nu/nu mice (6 weeks old on purchase) (Japan SLC Inc.) were used. The mice were acclimated using standard feeds and beddings and given food and drinking water ad libitum for 1 week before cancer cells were transplanted. At approximately 1 week before an imaging experiment, 1 x 10 6 Colon 26 mouse colon cancer cells (Riken, Japan) were subcutaneously injected into the right shoulder and the right thigh of each mouse, and 1 x 10 6 Colon 26 mouse colon cancer cells into which the HER2 gene was artificially transferred were subcutaneously injected into the left shoulder and the left thigh of each mouse. Tumor cells had been all established by the time of the experiment.
- mice The body weights of the mice were between 17 and 22 g. Then 200 microliters (13 nmol in terms of ICG) of HER2-bindig-molecule-immobilized ICG-HSA or ICG-HSA to which nothing was immobilized was intravenously injected into the tumor-bearing mice. The mice were euthanized with carbon dioxide 24 hours after administration. The tumor tissues were removed and transferred to plastic tubes. An aqueous solution of 1% Triton X-100 was added to each of the tubes in an amount 1.25 times the weight of the tumor tissues. Each of the resulting mixtures was homogenized with a plastic pestle.
- DMSO was added to each mixture in an amount 20.25 times the weight of the tumor tissues to prepare a solution of the dye extracted from the tumor tissues.
- the tumor tissues were removed from tumor-bearing mice into which HER2-binding-molecule-immobilized ICG-HSA was not administered.
- the tumor tissues were transferred to plastic tubes.
- An aqueous solution of 1% Triton X-100 was added to each of the tubes in an amount 1.25 times the weight of the tumor tissues.
- Each of the resulting mixtures was homogenized with a plastic pestle to prepare a solution of the tumor tissues in Triton-X100.
- a HER2-binding-molecule-immobilized ICG-HSA solution having a known concentration was diluted with the solution of the tumor tissues in Triton X-100 to various concentrations. Then DMSO was added to each of the resulting dilute solutions in an amount 20.25 times the amount of each dilute solution to prepare standard solutions for calibration. The fluorescence intensity of the solutions of the dye extracted from the tumor tissues and the standard solutions for calibration was measured with the solutions in the tubes using IVIS (registered trademark) Imaging System 200 Series (XENOGEN Corporation) to quantitatively determine the amount of the dye in the tumor tissues.
- IVIS registered trademark
- Imaging System 200 Series XENOGEN Corporation
- Table 11 summarizes the number of linkers immobilized to HER2-binding-molecule-immobilized ICG-HSA, the number of HER2-binding molecules, the binding affinity to HER2 in vitro, the accumulation in the Colon 26 tumor into which the HER2 gene was transferred, and the ratio of the accumulation in the Colon 26 tumor into which the HER2 gene was transferred to the accumulation in the wild-type Colon 26 tumor.
- HER2-binding-molecule-immobilized ICG-HSA samples except L11-ICG-HSA, although the accumulation was reduced, selective accumulation in the Colon 26 tumor into which the HER2 gene was transferred was observed with respect to the wild-type Colon 26 tumor, compared with ICG-HSA into which nothing was immobilized.
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Claims (19)
- Kontrastmittel für fotoakustische Bildgebung, umfassend:einen Komplex mit Albumin, das kovalent an mindestens einen organischen Farbstoff gebunden ist, der Licht mit einer Wellenlänge im nahen Infrarotbereich absorbiert,wobei der organische Farbstoff, der Licht mit einer Wellenlänge im nahen Infrarotbereich absorbiert, durch Formel (II) dargestellt ist:
B-B' (II)
wobei in der Formel (II) B die Formel (i) oder (ii) darstellt und B' eine der Formeln (iii) bis (vi) darstellt,wobei die "*" in den Formeln (i) und (ii) jeweils an B' von Formel (II) binden, und die "*" in den Formeln (iii) bis (vi) jeweils an B von Formel (II) binden:wobei in den Formeln (i) und (ii) die "Z" jeweils ein Wasserstoffatom, eine Sulfonsäuregruppe oder einen zyklischen aromatischen Ring darstellen, der aus der Gruppe ausgewählt ist, die aus einem Benz[e]indolring, einem Benz[f]indolring und einem Benz[g]indolring gemeinsam gebildet mit einem an ein entsprechendes der "Z" gebundenen Indolring, besteht; und die Wasserstoffatome des zyklischen aromatischen Rings jeweils durch eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen, eine Alkoxygruppe mit 1 bis 10 Kohlenstoffatomen, oder eine Sulfonsäuregruppe ersetzt sein können;wobei in den Formeln (i) und (ii) die R1 jeweils eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen oder -(CH2)b-SO3 - darstellen, wobei b eine ganze Zahl von 1 bis 10 darstellt; wobei ein Halogenidion oder ein Ion einer organischen Säure als ein Gegenion enthalten sein kann, wenn die R1s jeweils eine Alkylgruppe darstellen; und wobei die R2 und R3 jeweils unabhängig voneinander darstellen: ein Wasserstoffatom, eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen, eine Alkoxygruppe mit 1 bis 10 Kohlenstoffatomen, -(CH2)b-SO3-, wobei b eine ganze Zahl von 1 bis 10 darstellt, oder -(CH2)b-SO3X, wobei b eine ganze Zahl von 1 bis 10 darstellt und X Natrium, Kalium, Ammonium, Triethylammonium, Lysin oder Arginin darstellt;wobei in den Formeln (i) und (ii) die "a" jeweils eine ganze Zahl von 1 bis 10 darstellen, und die "n" jeweils 2 oder 3 darstellen; undwobei in der Formel (ii) R4 eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen oder -(CH2)b-SO3X darstellt, wobei b eine ganze Zahl von 1 bis 10 darstellt und X Natrium, Kalium, Ammonium, Triethylammonium, Lysin oder Arginin darstellt:dadurch gekennzeichnet, dass:
*-OH (vi)
im Kontrastmittel eine Anzahl kovalent an das Albumin des Komplexes gebundener Moleküle des organischen Farbstoffs durchschnittlich 1,6 oder mehr beträgt. - Kontrastmittel für fotoakustische Bildgebung, umfassend:einen Komplex, der Albumin enthält, welches kovalent an mindestens einen organischen Farbstoff gebunden ist, der Licht einer Wellenlänge im nahen Infrarotbereich absorbiert,wobei in der Formel (I) A eine Bindungsstelle von Albumin mit einer fehlenden Aminogruppe darstellt, A' der durch die unten beschriebene Formel (i') oder Formel (ii') dargestellte organische Farbstoff ist, und "*" in den Formeln (i') und (ii') jeweils an ein Stickstoffatom (N) in der Formel (I) gebunden ist:wobei in den Formeln (i') und (ii') die "Z" jeweils ein Wasserstoffatom, eine Sulfonsäuregruppe oder einen zyklischen aromatischen Ring darstellen, der aus der Gruppe ausgewählt ist, die aus einem Benz[e]indolring, einem Benz[f]indolring und einem Benz[g]indolring gemeinsam gebildet mit einem an ein entsprechendes der "Z" gebundenen Indolring, besteht; und die Wasserstoffatome des zyklischen aromatischen Rings jeweils durch eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen, eine Alkoxygruppe mit 1 bis 10 Kohlenstoffatomen, oder eine Sulfonsäuregruppe ersetzt sein können,wobei in den Formeln (i') und (ii') die R1 jeweils eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen oder -(CH2)b-SO3 - darstellen, wobei b eine ganze Zahl von 1 bis 10 darstellt; wobei ein Halogenidion oder ein Ion einer organischen Säure als Gegenion enthalten sein kann, wenn die R1 jeweils eine Alkylgruppe darstellen; und wobei die R2 und R3 jeweils unabhängig voneinander darstellen: ein Wasserstoffatom, eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen, eine Alkoxygruppe mit 1 bis 10 Kohlenstoffatomen, -(CH2)b-SO3 -, wobei b eine ganze Zahl von 1 bis 10 darstellt, oder -(CH2)b-SO3X, wobei b eine ganze Zahl von 1 bis 10 darstellt und X Natrium, Kalium, Ammonium, Triethylammonium, Lysin oder Arginin darstellt,wobei in den Formeln (i') und (ii') die "a" jeweils eine ganze Zahl von 1 bis 10 darstellen; und die "n" jeweils 2 oder 3 darstellen, undwobei in Formel (ii') R4 eine Alkylgruppe mit 1 bis 10 Kohlenstoffatomen oder -(CH2)b-SO3X darstellt, wobei b eine ganze Zahl von 1 bis 10 darstellt und X Natrium, Kalium, Ammonium, Triethylammonium; Lysin oder Arginin darstellt;dadurch gekennzeichnet, dass:
im Kontrastmittel eine Anzahl kovalent an das Albumin des Komplexes gebundener Moleküle des organischen Farbstoffs durchschnittlich 1,6 oder mehr beträgt. - Das Kontrastmittel für fotoakustische Bildgebung nach Anspruch 3, wobei Formel (i') durch eine der Formeln (i-1) bis (i-6) dargestellt ist:
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 6, wobei das Albumin menschliches Serumalbumin, eine Variante menschlichen Serumalbumins, ein Fragment menschlichen Serumalbumins, oder ein Fragment einer Variante menschlichen Serumalbumins ist.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 7, wobei der Komplex in Form von Partikeln vorliegt, und wobei die hydrodynamische mittlere Partikelgröße der Partikel, gemessen durch eine Dynamische-Lichtstreuungs-Methode, 200 nm oder weniger beträgt.
- Kontrastmittel für fotoakustische Bildgebung nach Anspruch 8, wobei die hydrodynamische mittlere Partikelgröße der Partikel, gemessen durch eine Dynamische-Lichtstreuungs-Methode, 50 nm oder weniger beträgt.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 9, ferner umfassend:
ein Fängermolekül. - Kontrastmittel für fotoakustische Bildgebung nach Anspruch 1, wobei der Komplex durch die Formel (III) dargestellt ist:
ALB-L-C (III)
wobei in der Formel (III), ALB das Albumin darstellt, das kovalent durch den organischen Farbstoff gebunden ist; C ein Fängermolekül darstellt; L eine Verbindungsgruppe darstellt, wobei ALB an L gebunden ist und L an C gebunden ist. - Kontrastmittel für fotoakustische Bildgebung nach Anspruch 10 oder 11, wobei das Fängermolekül ein Protein, ein Polypeptid oder ein Peptid ist.
- Kontrastmittel für fotoakustische Bildgebung nach Anspruch 12, wobei das Protein ein Einkettenantikörper ist.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 13, ferner umfassend einen Zusatzwirkstoff.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 14, wobei im Kontrastmittel eine Anzahl kovalent an das Albumin des Komplexes gebundener Moleküle des organischen Farbstoffs durchschnittlich 2,3 oder mehr beträgt.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 15, wobei im Kontrastmittel eine Anzahl kovalent an das Albumin des Komplexes gebundener Moleküle des organischen Farbstoffs durchschnittlich 3,1 oder weniger beträgt.
- Kontrastmittel für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 16, wobei im Kontrastmittel eine Anzahl kovalent an das Albumin des Komplexes gebundener Moleküle des organischen Farbstoffs durchschnittlich 3,0 oder weniger beträgt.
- Verwendung des Kontrastmittels für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 17 als Kontrastmittel für Tumorbildgebung.
- Verwendung des Kontrastmittels für fotoakustische Bildgebung nach einem der Ansprüche 1 bis 17 als Kontrastmittel für Lymphknotenbildgebung.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012161643 | 2012-07-20 | ||
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WO2014013732A1 (en) | 2012-07-20 | 2014-01-23 | Canon Kabushiki Kaisha | Compound and photoacoustic imaging contrast medium containing the compound |
US9592307B2 (en) | 2012-07-20 | 2017-03-14 | Canon Kabushiki Kaisha | Compound and photoacoustic imaging contrast medium containing the compound |
CN105008402B (zh) | 2013-02-22 | 2018-09-18 | 佳能株式会社 | 近红外色素结合的透明质酸衍生物和包括其的光学成像用造影剂 |
US10245329B2 (en) | 2014-09-08 | 2019-04-02 | Canon Kabushiki Kaisha | Composition having dye and conjugate of polyethyleneglycol and additive and contrast agent for photoacoustic imaging having the same |
JP6652808B2 (ja) | 2014-10-16 | 2020-02-26 | キヤノン株式会社 | 重合体、前記重合体を有する光音響イメージング用造影剤 |
JP6736278B2 (ja) | 2014-10-24 | 2020-08-05 | キヤノン株式会社 | 重合体、前記重合体を有する光音響イメージング用造影剤 |
JP6552236B2 (ja) | 2015-03-23 | 2019-07-31 | キヤノン株式会社 | 近赤外色素結合トランスフェリン、前記近赤外色素結合トランスフェリンを有する光音響イメージング用造影剤 |
KR20160142199A (ko) | 2015-06-02 | 2016-12-12 | 삼성메디슨 주식회사 | 광음향 영상화를 위한 조영 조성물 및 그를 사용한 광음향 영상화하는 방법 |
JP6789636B2 (ja) | 2015-09-30 | 2020-11-25 | キヤノン株式会社 | 重合体、前記重合体を有する光音響イメージング用造影剤 |
JP6752582B2 (ja) | 2016-02-08 | 2020-09-09 | キヤノン株式会社 | 光音響イメージング用造影剤 |
CN107022350A (zh) * | 2017-04-20 | 2017-08-08 | 深圳大学 | 一种荧光造影材料及其制备方法与应用 |
US20200054771A1 (en) * | 2018-08-17 | 2020-02-20 | Endra Life Sciences Inc. | Thermoacoustic imaging method and system and thermoacoustic imaging contrast agent |
CN110201189B (zh) * | 2019-06-03 | 2021-12-07 | 沈阳药科大学 | 白蛋白结合型近红外荧光染料-马来酰亚胺共轭物 |
JP2022548019A (ja) * | 2019-09-13 | 2022-11-16 | データレース リミテッド | 組成物 |
CN113149966B (zh) * | 2021-03-09 | 2022-11-18 | 中国药科大学 | Nir/pet双模态造影剂及其制备方法与应用 |
CN113149967B (zh) * | 2021-03-09 | 2022-11-08 | 中国药科大学 | Nir/mri双模态造影剂及其制备方法与应用 |
CN113004254B (zh) * | 2021-03-09 | 2022-08-05 | 中国药科大学 | 以吲哚氰绿衍生物为载体的配体及其制备方法与应用 |
CN114019155A (zh) * | 2021-10-25 | 2022-02-08 | 复旦大学 | 一种基于抗酪氨酸羟化酶抗体的免疫探针及其制备与应用 |
CN113861717A (zh) * | 2021-11-09 | 2021-12-31 | 西安康福诺生物科技有限公司 | 一种用于荧光标记的水溶性吲哚菁绿染料及其合成方法 |
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JP4098839B2 (ja) | 1995-01-30 | 2008-06-11 | 株式会社同仁化学研究所 | インドシアニン化合物 |
DE69632244T2 (de) | 1995-01-30 | 2005-04-14 | Daiichi Pure Chemicals Co. Ltd. | Diagnostisches Markierungsmittel |
WO2004024191A2 (en) * | 2002-09-10 | 2004-03-25 | University Of Maryland, Baltimore | Use of metallic particles to improve fluorescence imaging |
US20080308744A1 (en) | 2003-11-18 | 2008-12-18 | Beth Israel Deaconess Medical Center | Serum Albumin Conjugated to Fluorescent Substances for Imaging |
AU2006204045B2 (en) * | 2005-01-05 | 2010-10-14 | Board Of Regents, The University Of Texas System | Conjugates for dual imaging and radiochemotherapy: composition, manufacturing, and applications |
GB2449433B (en) * | 2007-05-21 | 2009-12-09 | Clairair Ltd | Optical gas sensor |
EP2305214B1 (de) | 2008-06-05 | 2018-01-03 | Shimadzu Corporation | Neue molekulare anordnung, molekulare sonde zur molekularen bildgebung und molekulare sonde für ein arzneiabgabesystem damit sowie molekulares bildgebungssystem und arzneiabgabesystem |
US8652441B2 (en) | 2009-10-05 | 2014-02-18 | Canon Kabushiki Kaisha | Contrast agent for photoacoustic imaging and photoacoustic imaging method |
MX2012005423A (es) | 2009-11-13 | 2012-06-14 | Merck Patent Gmbh | Anticuerpos anti-integrina unidos a nanoparticulas cargadas con agentes quimioterapeuticos. |
JP2011184329A (ja) | 2010-03-05 | 2011-09-22 | Canon Inc | 化合物及び前記化合物を有する粒子及び前記粒子を有する造影剤 |
US8491908B2 (en) | 2010-06-01 | 2013-07-23 | Canon Kabushiki Kaisha | Composite particle, contrast agent for photoacoustic imaging, and method for producing the composite particle |
WO2012026608A1 (en) | 2010-08-24 | 2012-03-01 | Canon Kabushiki Kaisha | Polymeric particle and hydrophilic dye having a sulfonate group encapsulated within the particle |
US8753608B2 (en) | 2010-08-24 | 2014-06-17 | Canon Kabushiki Kaisha | Complex and contrast agent for photoimaging using the same |
US9138492B2 (en) | 2012-02-23 | 2015-09-22 | Canon Kabushiki Kaisha | Particle containing hydrophobic dye having cyanine structure, and contrast agent containing the particle |
WO2014013732A1 (en) | 2012-07-20 | 2014-01-23 | Canon Kabushiki Kaisha | Compound and photoacoustic imaging contrast medium containing the compound |
US9592307B2 (en) | 2012-07-20 | 2017-03-14 | Canon Kabushiki Kaisha | Compound and photoacoustic imaging contrast medium containing the compound |
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CN104487098A (zh) | 2015-04-01 |
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EP2861264A1 (de) | 2015-04-22 |
US9675715B2 (en) | 2017-06-13 |
WO2014013729A1 (en) | 2014-01-23 |
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